Over the past few years, heterologous expression systems have often been used to study the expression as well as the pharmacological and biochemical characteristics of membrane receptors.
Although a significant expression can be obtained in some expression systems in mammalian cells, there have been problems, in particular in the case of some types of receptors such as the G-protein-coupled receptors.
The G-protein-coupled receptors belong to the superfamily of receptors with seven transmembrane domains. They comprise, for example, the adrenergic or muscarinic receptors, and all have the same structure which is made up of a polypeptide chain comprising seven hydrophobic domains which cross the membrane lipid bilayer.
When it is desired to express these receptors in mammalian cell systems, a relatively low density of receptors expressed by the said cells, rarely exceeding a few picomoles of receptor per milligram of membrane protein, is generally obtained. Although these levels of expression are sufficient for a functional and pharmacological characterization, they clearly limit the type of biochemical, biophysical and structural studies which can be carried out. A fortiori, this expression system cannot be used for the production of receptors in a large quantity, for example for their therapeutic use.
To increase the quantity of receptors obtained, various teams have sought to produce them in a baculovirus/insect cell system; in many cases, baculoviruses expressing G-protein-coupled receptors have been able to produce these recombinant receptors in cells of the Spodoptera frugiperda Sf9 or Sf21 lines, up to levels reaching 30 to 100 picomoles per milligram of membrane protein. These systems have made it possible to make significant progress in the study of the palmitoylation of receptors and also to study the effects produced by various agonists and antagonists, or to carry out the reconstitution of artificial receptors.
However, the baculovirus/insect cell system has the major disadvantage of expressing a high proportion of inactive receptors. The receptors, which are recovered in the membrane fraction of the cells infected with the baculoviruses are in an immature and incompletely glycosylated form. This probably results from a saturation of the normal post-translational maturation pathway, which brings about the retention of immature receptors in the membranes of the endoplasmic reticulum or in the Golgi apparatus. To obtain functional receptors, it is necessary in this case to include a purification step based on the biological activity of the receptor (for example an affinity chromatography step).
It would therefore be necessary to develop a system which makes it possible to easily separate the plasma membrane comprising the mature receptors from the other membrane fractions such as the endoplasmic reticulum or the membranes of the Golgi apparatus which comprise the biologically inactive, immature receptor.